Supplement ? Tubulin microtentacles in detached mammary epithelial cells. This project focuses on metastasis of circulating breast tumor cells via unique microtentacles (McTNs) that were first discovered in the PI?s laboratory. The original renewal application (R01-CA124704-06A1) received a 2nd percentile score, but it was suggested that the project could be even further strengthened by incorporating studies that directly test McTN-dependent reattachment of circulating tumor cells (CTCs) under the complex conditions of in vivo blood flow. We have recently developed a highly productive collaboration with Dr. Norann Zaghloul to image CTC metastasis in zebrafish with exceptionally high-speed spinning disk confocal microscopy (200 frames per second). The proposed administrative supplement would support this collaboration to use in vivo imaging in zebrafish to study how the basic molecular mechanisms of R01- CA124704 impact CTC metastasis via McTN function. In the following specific aims, we will test the hypothesis that genetic or therapeutic dysregulation of the actin cortex induces tubulin McTNs that promote the metastatic reattachment of circulating tumor cells, with unprecedented resolution under conditions of in vivo bloodflow. The supplemental aims will address the 3 molecular mechanisms originally targeted in R01-CA124704 (PTEN-PI3K, Src, ROCK), but will now allow precise resolution of the initial steps of CTC reattachment during metastasis. Specific Aim 1: Target pathways activated by PTEN loss to influence CTC reattachment during metastasis. A) Test reattachment efficiency of MCF10A cells with defined genetic mutations in PTEN and K-Ras. B) Compare effects of PI3K inhibition (BKM120) and MEK inhibition (U0126) on CTC reattachment. Specific Aim 2: Test the role of Src inhibition on CTC reattachment efficiency. A) Examine CTC reattachment of human MDA-231 breast tumor cells with active and inactive Src mutations. B) Determine the impact of chemical Src inhibition (Dasatinib) on CTC reattachment. Specific Aim 3: Examine how targeting Rho/ROCK mediated actin contractility impacts CTC reattachment. A) Alter Rho/ROCK-mediated actin contractility with active/inactive Myosin mutations in Bt-549 tumor cells. B) Test effects of ROCK inhibition with Y-27632 on CTC reattachment. Compounds targeting these actin mechanisms (PI3K, MEK, Src, ROCK) are the subject of more 600 cancer clinical trials (www.clinicaltrials.gov), so the findings on CTC reattachment could have a wide impact on the field, as outlined recently by the PI? s lab in Clinical Cancer Research.